The Ca2+ signal observed in individual fura-2-loaded hepatocytes stimulated with the alpha 1-adrenergic agonist phenylephrine consisted of a variable latency period, a rapid biphasic increase in the cytosolic free Ca2+, followed by a period of maintained elevated cytosolic Ca2+ (plateau phase) that depended on the continued presence of both agonist and external Ca2+. Microinjection of guanosine-5'-O-(3-thiophosphate) elicited a Ca2+ transient with the same basic features. The Ca2+ transient resulting from microinjecting inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) occurred with essentially no latency period and consisted of a rapid spike that decayed back to preinjection levels within 15 s. Microinjection of inositol 1,4,5-trisphosphorothioate (thio-IP3), a nonmetabolizable analog of Ins-1,4,5-P3, elicited a Ca2+ transient that was initially identical to that observed with Ins-1,4,5-P3, except that the cytosolic Ca2+ remained elevated. The maintained thio-IP3-induced Ca2+ increase was dependent on the presence of external Ca2+, suggesting an activation of Ca2+ influx. Reintroduction of external Ca2+ in the presence of 5 microM phenylephrine to Ca(2+)-depleted cells resulted in a 2-fold greater rate of rise in the cytosolic Ca2+ compared to the rate observed upon Ca2+ addition to cells Ca(2+)-depleted by preatement with thapsigargin. The rate of Ca2+ rise upon Ca2+ addition to cells microinjected with thio-IP3 was similar to that observed with phenylephrine. Coinjection of the cells with thio-IP3 plus heparin reduced the rate of Ca2+ rise upon Ca2+ addition to that observed in thapsigargin-treated cells. These data indicate that the mechanism responsible for receptor-mediated stimulation of Ca2+ entry into hepatocytes involves not only capacitative Ca2+ entry but also an additional component mediated directly by Ins-1,4,5-P3.